The cytochrome P450-mixed function oxidase system play a crucial role in the metabolism of many endogenous and exogenous compounds P450 enzymes catalyze the metabolic detoxification of chemical carcinogens and toxins as well as the metabolic activation of certain compounds to highly reactive intermediates. Our interests are in identifying the specific peptides and amino acid residues in the P450 2B1 active site by labeling the substrate binding site with several structurally distinct mechanism-based inactivators. Since P450s are membrane proteins and the peptides derived from P450s are generally very hydrophobic, conventional enzymatic digestion, HPLC separation and gas-phase N-terminal sequencing has not been successful. MALDI-MS analysis makes it possible for us to analyze the entire cyanogen bromide digest of P450 2B1 and to detect and identify the uniquely labeled peptide. In addition, sequence information on the modified peptide(s) can be obtained by analysis of the metastable decay fragment of MALDI ions formed during post-source decay. Our studies on the characterization of the mechanism-based inactivation of P450 2B1 by benzyl-1-aminobenzotriazole (BBT) and the identification of five products that were generated from BBT metabolism. MALDI-MS has also been used to identify heme adducts formed from a series of acetylinic inhibitors of P450s.